Colorimetric determination of dehydrogenases

ABSTRACT

Test composition and method for assaying dehydrogenase enzymes in test fluids which comprises contacting the fluid with a test reagent comprising a coenzyme, a substrate for the dehydrogenase enzyme, a terminal acceptor dye and Meldola&#39;&#39;s Blue as an intermediary hydrogen carrier. A preferable embodiment of the present invention comprises incorporating the test composition with a solid carrier therefor and using the same as a dip and read test device.

United States Patent 1191 Fosker et al.

1 1 May 8, 1973 [54] COLORIMETRIC DETERMINATION OF DEHYDROGENASES [75] Inventors: Alan Phillip Fosker, High Wycombe; Patrick James Mill,

Beaconsfield, both of England [73] Assignee: Miles Laboratories Inc., Elkhart,

Ind. 1

22 Filed: Sept. 16, 1971 21 Appl.No.: 181,192

[30] Foreign Application Priority Data Sept. '17, 1970 Great Britain .J ..44,498/70 [52] US. Cl. ..]l95/l03.5 R, 195/99, 23/253 TP [51] Int. Cl. .l ..G0ln 31/14 [58] Field of Search ..l95/103.5 R;

[56] References Cited OTHER PUBLICATIONS Anal. Abstracts 17:2541

Colswick et al., Methods in Enzymology, Vol. IV, p. 329-336, (1957).

Primary Examiner-Alvin E. Tanenholtz Assistant ExaminerMax D. Hensley Att0rney-Joseph C. Schwalbach et al.

[57] ABSTRACT 8 Claims, N0 Drawings COLORIMETRIC DETERMINATION OF DEHYDROGENASES BACKGROUND OF THE INVENTION The determination of certain dehydrogenases linked to the coenzymes nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) is of medical importance in the diagnosis of several diseases. For example, an elevated value of lactic dehydrogenase (LDH) can be indicative of myocardial infarction, convulsive disorder, carcinoma of the prostate gland or hepatic disease. A rise in 6-phosphogluconate dehydrogenase (6-PGD) in vaginal fluid can be indicative of trichomonas vaginitis or gynecologic cancer. A deficiency of glucose-6-phosphate dehydrogenase (G6-PD) can indicate hereditary as well as drug induced haemolytic anaemia.

DESCRIPTION OF THE PRIOR ART The coenzyme-linked dehydrogenases referred to above are known to transfer hydrogen atoms from their substrates to the coenzymes NAD and NADP. The reduction of the coenzyme is usually followed by a change in optical density of 340 millimicrons. However, this wavelength is in the ultra-violet region so that complex and expensive apparatus is required to measure such changes. A colorimetric method uses smaller concentrations of coenzymes but adds a terminal acceptor system which will be reduced by the reduced .coenzyme. Thus, the coenzyme is restored to its original oxidized state, ready to accept more hydrogen from the substrate, and the terminal acceptor system is so chosen that its reduction produces a color change which is proportional to the enzyme activity.

No single substance is known which will both couple with reduced coenzyme and give a good color change on reduction. In the past, both a terminal acceptor dye which gives the color change and an intermediary carrier have been used in dehydrogenases. In such methods, the carrier is reduced by the reduced coenzyme, and in turn reduces the terminal acceptor. Typical acceptors include 2-(piodophenyl)- 3-(p-nitrophenyl)-5-phenyl tetrazolium chloride, (hereinafter referred to as tetrazolium salt) and the corresponding bromide and iodide and 2,6- dichloroindophenol. Only phenazine methosulfate has previously been found suitable as an intermediary hydrogen carrier, but it is unstable and cannot beused for prepackaged reagent kits.

SUMMARY OF THE INVENTION We have now found that Meldolas Blue, which is sufficiently stable to be used in prepackaged kits, is a suitable intermediary hydrogen carrier for this purpose. More particularly we have found that the enzymes LDH, 6-PGD and G6 PD can be assayed colorimetrically using a buffered aqueous mixture consisting of Meldolas Blue, tetrazolium salt (as defined above) as a terminal acceptor dye, a substrate for the enzyme, and NAD or NADP as coenzyme, or by use of test material comprising a carrier member such as a bibulous paper strip containing or carrying such a solution. When such a composition or material comes into contact with one of said enzymes, a color change occurs which can be the assay of variousfollowed visually or by means of a spectrophotometer, and which, when compared with standards containing known amounts of the enzyme to be determined, serves as a measure of the enzyme concentration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The reactions which occur in the present invention can be illustrated as follows:

1. -phospho-d-gluconate NAPD 2 6-phospho-2- keto-d-gluconate NADPI-l C0 2. d-glucose-fi-phosphate NADP d-glucono-S- lactone-6-phosphate NADPH 3. lactic acid NAD 2 pyruvic acid NADl-l Meldolas Blue is an oxazine dye known chemically as 9-dimethylaminobenzo-oz-phenazoxonium chloride, first discovered in l879 by Meldola, and having the formula In one aspect therefore the invention comprises a composition for use in the colorimetric assay of a dehydrogenase enzyme, which comprises a substrate for the said enzyme, as coenzyme NAD or NADP, a terminal acceptor dye, and Meldolas Blue as an intermediary hydrogen carrier.

The composition may be in the form of a buffered aqueous solution. Preferably however the composition is deposited on or incorporated in a carrier, and the product is used as a dip and read test device. Such a test device can be readily made by impregnating a bibulous material with a solution of the test composition, and subsequently drying it, or by impregnating the bibulous material successively with the various ingredients of the test composition, either singly or two or more at a time, and drying it after each such impregnation. Alternatively, a finely divided, dry, intimate mixture of the ingredients can be adhesively attached to the surface of the carrier. Another embodiment involves incorporating the test composition with a polymeric semi-permeable membrane and either using this membrane as such or attaching the membrane to a carrier. The carrier material may for example be filter paper, wood strip, synthetic plastic or non-woven or woven fabric, filter paper having a thickness of from 0.01 inch to 0.02 inch being generally preferred. Such test devices constitute a further aspect of the invention.

While the preferred terminal acceptor dye is a tetrazolium salt, other compounds can be used, including other tetrazolium dyes, and 2,6-dichloroindophenol. The substrate will of course correspond to the particular dehydrogenase to be determined; thus for determining LDH, lactic acid will be used; for determining 6-PGD, o-phosphogluconate; and for determining G6-PD, glucose-6-phosphate. The coenzyme is NAD or NADP, depending on the coenzyme specificity of the enzyme to be measured.

Another aspect the invention consists in a method for the assay of a dehydrogenase enzyme, which comprises contacting a composition comprising the said enzyme with a predetermined quantity of a composition as described above in the form of a buffered aqueous solution, or preferably with a test material comprising a carrier impregnated with or carrying on its surface such a composition, and comparing the color developed in the solution, or on the test material after it has been removed from contact with the composition containing the enzyme, with a standard color chart.

The following examples illustrate the invention.

EXAMPLE 1 A mixture for use in the colorimetric assay of G6-PD was prepared from the following ingredients:

0.3 ml of 0.1 molar aqueous sodium phosphate solution (pH 7.5)

0.2 ml. of 0.1 molar aqueous magnesium chloride solution 0.1 ml. of an aqueous solution containing 1.4 millimoles of NADP 0.1 ml. of an aqueous solution containing 0.3 percent gelatin 0.2 ml. ofa aqueous solution containing 3 mg. of disodium glucose-o-phosphate per ml. of water 0.3 ml. of an aqueous solution containing 5 mg. of

tetrazolium salt per ml. of water 0.05 ml. of an aqueous solution containing 1 mg. of

Meldolas Blue per ml. of water 1.7 ml. ofwater To this mixture was added 0.05 ml. of an aqueous solution containing 0.03 mg. of G6-PD per ml. of water.

The resulting mixture was placed in a cuvette and the change in optical density at 500 millimicrons was followed with a spectrophotometer or colorimeter at 25C. The rate of change in optical density was linear for 5 minutes and was taken as a measure of the enzyme activity.

EXAMPLE 2 A mixture was prepared having the same composition as that described in Example 1 except that 6- phosphogluconate replaced the glucose-6-phosphate as a substrate. 6-PGD was determined colorimetrically by observing the rate of change in optical density with a spectrophotometer or colorimeter at 500 millimicrons and comparing the results with a standard obtained by carrying out the test procedure with solutions containing known amounts of 6-PGD. As in Example 1, the rate of change in optical density was linear for about 5 minutes.

EXAMPLE 3 A composition for use in the colorimetric assay of LDH was prepared by mixing the following components:

1.10 ml. of an aqueous borate buffer solution containing 3.10 g. boric and 1.67 g. borax per 100 ml. water (pH8.6)

0.25 ml. of 0.5 molar aqueous sodium lactate solution 0.10 ml. of an aqueous solution containing 0.3 percent gelatin 0.50 ml. of an aqueous solution containing 1 mg. of

NAD per ml. of water 0.30 ml. of aqueous solution containing 5 mg. of

tetrazolium salt per ml. of water 0.05 ml. of an aqueous solution containing 1 mg. of

Meldolas Blue per ml. of water 0.50 ml. ofwater To this mixture was added 0.20 ml. of human serum. When the resulting composition was placed in a spectrophotometer, a linear change of optical density was observed for a period of 10 minutes at a rate of 0.183 optical density units per minute at a wavelength of 500 millimicrons.

EXAMPLE 4 A strip of Whatman No. filter paper about 1 centimeter wide and 14 centimeters long was impregnated successively with the following solutions and dried in a stream of warm air between each application:

1. 0.05 molar sodium phosphate in 50 percent aqueous methanol buffered to a pH of 7.5

2. 0.05 molar magnesium chloride in 50 percent aqueous methanol 3. an aqueous solution containing 3 mg. of disodium glucose--phosphate per ml. of water.

4. an aqueous solution containing 1.4 millimoles of NADP 5. a methanol solution containing 0.04 mg. of Meldola's Blue and 5 mg. of tetrazolium salt per ml. of methanol.

When 0.05 ml. of aqueous solutions containing respectively 0.005 0.10 mg. of G6-PD per ml. of water were applied to the strip thus prepared, the original pale blue color of the strip changed to a color ranging from a pinkish red to a deep reddish purple dependent upon the G6-PD enzyme concentration. The strips can be stored for up to 18 months in the dark in the presence of silica gel without loss of activity.

EXAMPLE 5 A strip of filter paper was impregnated with the same solutions as in Example 4 except that 6-phosphogluconate replaced the glucose-6-phosphate. When the strip was contacted with an aqueous solution of 6-PGD, a color change occurred which was indicative of the amount of 6-PGD enzyme present.

EXAMPLE 6 A paper was impregnated as described in Example 4 with the following solutions and used to assay LDH:

l. 1.5 molar aqueous sodium borate solution 2. 1.5 molar aqueous sodium lactate solution 3. an aqueous solution containing 2 mg. of NAD per ml. of water 4. a methanol solution containing 0.04 mg. of Meldola's Blue and 5 mg. of tetrazolium salt per ml. of methanol.

When a paper strip so prepared was contacted with a preparation of beef heart lactic dehydrogenase, the colors produced by solutions containing 0, 80, 160, 240, 800 and 3,200 international units of LDH activity could be readily distinguished.

What is claimed is:

1. A composition for the determination of a dehydrogenase enzyme in a test fluid which comprises a coenzyme selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, a corresponding substrate for said enzyme, a terminal acceptor dye and Meldolas Blue as an intermediary hydrogen carrier.

2. A composition according to claim 1 wherein the terminal acceptor dye is selected from the group consisting of Z-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium bromide and 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium iodide.

3. A composition according to claim 1 wherein the enzyme beingvdetermined is lactic dehydrogenase and the substrate is lactic acid.

4. A composition according to claim 1 wherein the enzyme being determined is glucose-6-phosphate dehydrogenase and the substrate is glucose-6- phosphate.

5. A composition according to claim 1 wherein the enzyme being determined is 6-phosphogluconate dehydrogenase and the substrate is 6-phosphogluconate.

6. A test device for the determination of a dehydrogenase enzyme comprising a carrier with which is incorporated the composition of claim 1.

7. A test device according to claim 6 wherein the carrier is a bibulous paper.

8. A method for the determination of a dehydrogenase enzyme, which comprises contacting the fluid containing the enzyme with a predetermined proportion of a composition comprising a coenzyme selected from the group consisting of dinucleotide and nicotinamide adenine dinucleotide phosphate, a corresponding substrate for said enzyme, a terminal acceptor dye and Meldolas Blue as an intermediary hydrogen carrier. 

2. A composition according to claim 1 wherein the terminal acceptor dye is selected from the group consisting of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium bromide and 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium iodide.
 3. A composition according to claim 1 wherein the enzyme being determined is lactic dehydrogenase and the substrate is lactic acid.
 4. A composition according to claim 1 wherein the enzyme being determined is glucose-6-phosphate dehydrogenase and the substrate is glucose-6-phosphate.
 5. A composition according to claim 1 wherein the enzyme being determined is 6-phosphogluconate dehydrogenase and the substrate is 6-phosphogluconate.
 6. A test device for the determination of a dehydrogenase enzyme comprising a carrier with which is incorporated the composition of claim
 1. 7. A test device according to claim 6 wherein the carrier is a bibulous paper.
 8. A method for the determination of a dehydrogenase enzyme, which comprises contacting the fluid containing the enzyme with a predetermined proportion of a composition comprising a coenzyme selected from the group consisting of dinucleotide and nicotinamide adenine dinucleotide phosphate, a corresponding substrate for said enzyme, a terminal acceptor dye and Meldola''s Blue as an intermediary hydrogen carrier. 